RB-1 was the first tumor suppressor gene to be identified and it has served as a prototype for this class of cancer-related genes. Genetic studies show that pRB is a multifunctional protein and over eighty different cellular pRN-related proteins have been reported. However, a detailed analysis of this protein has been difficult because most of its properties require a common domain, the pocket domain, and this domain has been refractory to structure/function studies. The recently published crystal structure of the pRN pocket has provided the information needed to specifically mutate conserved residues on the surface of pRB, without disrupting the overall structure of the pocket domain. These studies are needed because of the multiple activities, and the large number of pRB-binding proteins, that depend on this domain. Currently it is unclear which interactions are needed for specific functions of pRB, and it is uncertain which functions of pRB are most relevant for tumor suppression. In the last cycle of this grant they have used the crystal structure to begin to dissect the functions of the pRB pocket domain. A panel of mutants has been prepared that disrupt the LXCXE-binding cleft of pRB. This structure is used by multiple viral oncoproteins to bind to pRB, and sequences similar to the viral LXCXE motif have been found in at least 21 of the cellular pRB-binding proteins reported to date. The LXCXE-binding cleft is maintained in all pRB-homologs that have been identified, suggesting that it is critical for a conserved function of pRB.